The present invention includes novel human E3xcex1 ubiquitin ligase polypeptides (huE3xcex1I and huE3xcex1II) and nucleic acid molecules encoding the same. The invention also relates to vectors, host cells, selective binding agents, such as antibodies, and methods for producing huE3xcex1 polypeptides. Also provided for are methods for the diagnosis, treatment, amelioration and/or prevention of diseases associated with huE3xcex1 polypeptides, as well as methods for identifying modulators of huE3xcex1 ligase activity.
Technical advances in the identification, cloning, expression and manipulation of nucleic acid molecules and deciphering of the human genome have greatly accelerated the discovery of novel therapeutics based upon deciphering of the human genome. Rapid nucleic acid sequencing techniques can now generate sequence information at unprecedented rates and, coupled with computational analyses, allow the assembly of overlapping sequences into the partial and entire genomes as well as the identification of polypeptide-encoding regions. A comparison of a predicted amino acid sequence against a database compilation of known amino acid sequences can allow one to determine the extent of homology to previously identified sequences and/or structural landmarks. The cloning and expression of a polypeptide-encoding region of a nucleic acid molecule provides a polypeptide product for structural and functional analyses. The manipulation of nucleic acid molecules and encoded polypeptides to create variants and derivatives thereof may confer advantageous properties on a product for use as a therapeutic.
In spite of significant technical advances in genome research over the past decade, the potential for the development of novel therapeutics based on the human genome is still largely unrealized. Many genes encoding potentially beneficial polypeptide therapeutics, or those encoding polypeptides which may act as xe2x80x9ctargetsxe2x80x9d for therapeutic molecules, have still not been identified. In addition, structural and functional analyses of polypeptide products from many human genes have not been undertaken.
Accordingly, it is an object of the invention to identify novel polypeptides and nucleic acid molecules encoding the same which have diagnostic or therapeutic benefit.
Most types of intracellular proteins are degraded through the ubiquitin-proteosome pathway. In this system, proteins are marked for protesomal degradation by the conjugation of ubiquitin molecules to the protein. Conjugation of the ubiquitin molecule initially involves activation by the E1 enzyme. Upon activation the ubiquitin molecule is transferred to the E2 enzyme which serves as a carrier-protein. The E2 enzyme interacts with a specific E3 ligase family member. The E3 ligase binds to proteins targeted for degradation and catalyzes the transfer of ubiquitin from the E2 carrier enzyme to the target protein. Since the target protein binds to the ligase prior to conjugatin, E3 ligase is the rate limiting step for ubiquitin conjugation and determines the specificity of the system. The ubiquitin chain serves as a degradation marker for the 26S proteosome (See Ciechanover, EMBO J., 17: 7151-7160, 1998).
There are only a few known E3 ligases and the sequence homology between them is low. The E3xcex1 family is the main family of intracellular ubiquitin ligases and is involved in N-end rule pathway of protein degradation. The N-end rule states that there is a strong relation between the in vivo half-life of a protein and the identity of its N-terminal amino acids. Accordingly, E3xcex1 enzyme binds directly to the primary destabilizing N-terminal amino acid and catalyzes ubiquitin conjugation thereby targeting the protein for degradation. E3xcex1 family members also recognize non-N-end rule substrates (See Ciechanover, EMBO J., 17: 7151-7160, 1998).
The E3xcex1 enzyme family currently consists of intracellular enzymes isolated from rabbit (Reiss and Hershiko, J. Biol. Chem. 265: 3685-3690, 1990), mouse (Kwon et al., Proc. Natl. Acad. Sci., U.S.A 95: 7898-7903, 1999), yeast (Bartel et al., EMBO J., 9: 3179-3189, 1990) and the C. elegans (Wilson et al., Nature, 368: 32-38, 1994; Genebank Accession No. U88308) counterparts termed UBR-1. Comparison of these known sequences indicates regions of high similarity regions (I-V) which suggest the existence of a distinct family. The regions of similarity contain essential residues for the recognition of N-end rule substrates. In region 1, the residues Cys-145, Val-146, Gly-173, and Asp-176 are known to be necessary for type-1 substrate binding in yeast and are conserved in the mouse. In regions II and III, residues Asp-318, His-321, and Glu-560 are essential for type-2 substrate binding in yeast and are also conserved in the mouse. In addition, there is a conserved zinc-finger domain in region I and a conserved RING-H2 domain in region IV (Kwon et al., Proc. Natl. Acad. Sci., U.S.A, 95: 7898-7903, 1999).
The full length mouse E3xcex1 cDNA sequence and a partial human E3xcex1 nucleotide sequence (≈1 kb) have recently been cloned and characterized as described in U.S. Pat. No. 5,861,312 and Kwon et al (Proc. Natl. Acad. U.S.A., 95: 7898-7908, 1999). The full length mouse E3xcex1 cDNA sequence is 5271 bp in length and encodes a 1757 amino acid polypeptide. The mouse E3xcex1 gene is localized to the central region of chromosome 2 and is highly expressed in skeletal muscle, heart and brain. The partial human E3xcex1 sequence was used to characterize tissue expression and chromosomal localization. This analysis indicated that the human E3xcex1 gene is located on chromosome 15q and exhibits a similar expression pattern as mouse E3xcex1 with high expression in skeletal muscle, heart and brain. As described herein, the present invention discloses two novel, full length, human E3xcex1 sequences (huE3xcex1I and huE3xcex1II) and a novel, full length mouse E3xcex1 sequence (muE3xcex1II). Expression of huE3xcex1I and huE3xcex1II mRNA is highly enriched in skeletal muscle tissues. Functionally, huE3xcex1 polypeptides are intracellular enzymes that control protein conjugation and degradation.
Increased proteolysis through the ubiquitin-proteosome pathway has been determined to be a major cause of rapid muscle wasting in many pathological states including but not limited to fasting, metabolic acidosis, muscle denervation, kidney failure, renal cachexia, uremia, diabetes mellitus, sepsis, AIDS wasting syndrome, cancer cachexia, negative nitrogen balance cachexia, bums and Cushing""s syndrome (See Mitch and Goldberg, New England J. Med, 335: 1897-1905, 1996). Studies in animal models have shown that muscle wasting disorders are associated with increased ubiquitin content in muscles, increased levels of mRNA transcripts encoding ubiquitin, E2 enzyme and proteosome subunit mRNA, and increased ubiquitin-conjugation to muscle-proteins (See Lecker et al. J. Nutr., 129: 227S-237S, 1999). In this context, the N-end rule pathway has been shown to play a role in muscle atrophy. E3xcex1 inhibitors, such as dipepetides and methyl ester, reduce the level of ubiquitin conjugation in atrophying rat muscles caused by sepsis, fasting and cancer cachexia (Soloman et al., Proc. Natl. Acad. Sci. U.S.A. 95: 12602-12607, 1999). These observations indicate that E3xcex1 plays a role in the overall increase in ubiquitination that is associated with and may mediate muscle atrophy in catabolic and other disease states.
Thus, identification of members of the N-end rule protein degradation pathway has led to a better understanding of protein degradation in human cells and the mechanisms of protein degradation in pathological condition which involve muscle atrophy. Identification of the two novel human E3xcex1 ubiquitin ligase genes and polypeptides, as described herein, will further clarify the understanding of these processes and facilitate the development of therapies for pathological conditions which involve abnormal or excessive protein degradation including conditions which involve atrophy of muscle.
The present invention relates to novel human E3xcex1 nucleic acid molecules and polypeptides encoded by these nucleic acid molecules.
The invention provides isolated nucleic acid molecules comprising or consisting of a nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence as set forth in SEQ ID NOS: 1 or 3;
b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NOS: 2 and 4;
f) a nucleotide sequence which hybridizes under moderate or highly stringent conditions to the compliments of (a) or (b); and
d) a nucleotide complementary to (a)-(c)
The invention also provides isolated nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the polypeptide set forth in SEQ ID NOS: 2 or 4, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4 and the percent identity for these nucleic acid sequences are determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm;
b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in SEQ ID NOS: 1 or 3;
c) the nucleotide sequence of the DNA insert in ATCC Deposit No. PTA-1489 or PTA-1490;
d) a nucleotide sequence of SEQ ID NOS: 1; 3; (a); or (b) encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
e) a nucleotide sequence of SEQ ID NOS: 1, 3, or (a)-(c) comprising a fragment of at least about 16 nucleotides;
f) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(e); and
g) a nucleotide sequence complementary to any of (a)-(d).
The invention also provides isolated nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NOS: 2 or 4 with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
b) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NOS: 2 or 4 with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
c) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NOS: 2 or 4 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
d) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NOS: 2 or 4 which has a C- and/or N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
e) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NOS: 2 or 4 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
f) a nucleotide sequence of(a)-(e) comprising a fragment of at least about 16 nucleotides;
g) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(f); and
h) a nucleotide sequence complementary to any of (a)-(e).
The invention also provides isolated polypeptides comprising the amino acid sequence selected from the group consisting of:
a) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4;
b) the mature amino acid sequence as set forth in SEQ ID NOS: 2 or 4 comprising a mature amino terminus at residues 1, and optionally further comprising an amino terminal methionine;
c) an amino acid sequence that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the amino acid sequence of the polypeptide of SEQ ID NOS: 2 or 4 wherein the polypeptide has an activity of the polypeptide se t forth in SEQ ID NOS: 2 or 4 and the percent identity for these amino acid sequences are determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm.
d) a fragment of the amino acid sequence set forth in SEQ ID NOS: 2 or 4 comprising at least about 25, 50, 75, 100, or greater than 100 amino acid residues, wherein the fragment has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
e) the amino acid sequence encoded by the DNA insert of ATCC Deposit No. PTA-1489 o r PTA-1490;
f) an amino acid sequence for an ortholog of SEQ ID NOS: 2 or 4; including the murine ortholog set out as SEQ ID NO: 6.
g) an allelic variant or splice variant of (a), (b), (e) or (f);
The present invention also provides isolated polypeptides comprising the amino acid sequence selected from the group consisting of:
a) the amino acid as sequence set forth in SEQ ID NOS: 2 or 4 with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
b) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4 with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
c) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4;
d) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4 which has a C- and/or N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4; and
e) the amino acid sequence as set forth in SEQ ID NOS: 2 or 4, with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NOS: 2 or 4.
The present invention provides expression vectors comprising the nucleic acid molecules set forth herein, host cells comprising the expression vectors of the invention, and a method of producing a human E3xcex1 polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced. An another embodiment provides for viral vectors comprising the nucleic acid molecules of the inventions. Further provided is a process for determining whether a compound inhibits huE3xcex1 polypeptide activity or production comprising exposing a host cell expressing huE3xcex1 polypeptide to the compound, and measuring huE3xcex1 polypeptide activity or production in said cell.
A transgenic non-human animal comprising a nucleic acid molecule encoding a huE3xcex1 polypeptide is also encompassed by the invention. The huE3xcex1 nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of a huE3xcex1 polypeptide, which may include increased circulating levels. The transgenic non-human animal is preferably a mammal, and more preferably a rodent, such as a rat or a mouse.
Also provided are derivatives of the huE3xcex1 polypeptides of the present invention, fusion polypeptides comprising the huE3xcex1 polypeptides of the invention, and selective binding agents such as antibodies capable of specifically binding the polypeptides of the invention.
Pharmaceutical compositions comprising the nucleotides, polypeptides, or selective binding agents of the present invention and a carrier, adjuvant, solubilizer, stabilizer, anti-oxidant, or other pharmaceutically acceptable formulation agent are also encompassed by the invention. The pharmaceutical compositions include therapeutically effective amounts of the nucleotides or polypeptides of the present invention, and involve methods of using the polypeptides and nucleic acid molecules.
The huE3xcex1 polypeptides and nucleic acid molecules of the present invention may be used for therapeutic or diagnostic purposes to treat, prevent, and/or detect diseases or disorders, including those recited herein.
Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of a huE3xcex1 polypeptide are also encompassed by the invention. One method comprises administering to an animal a nucleic acid molecule encoding a huE3xcex1 polypeptide. In another method, a nucleic acid molecule comprising elements that regulate or modulate the expression of a huE3xcex1 polypeptide may be administered. Examples of these methods include gene therapy, cell therapy and antisense therapy as further described herein. Further provided is a method of identifying a compound which binds to a huE3xcex1 polypeptide comprising.
A device, comprising a membrane suitable for implantation and host cells expressing a huE3xcex1 polypeptide encapsulated within said membrane, wherein said membrane is permeable to said protein product and impermeable to materials detrimental to said cells is also encompassed by the present invention.